Western Blot

2.7.1. Protein extraction

After treatments, plates were removed from the incubator and put on ice to keep the temperature as close to 4°C as possible to inhibit any enzymatic activity that might cause protein degradation (proteases) or dephosphorylation of phosphatases. Media were disposed of and cells were carefully washed twice with 1 mL of cold PBS. Then, whole-cell lysates were prepared by extraction in 200 µL/well of radioimmuno- precipitation assay (RIPA) lysis buffer (10 mM Tris-HCl, pH 7.4; 150 mM NaCl; 0.1% SDS; 1% Triton-X100; 1% Sodium deoxycholate; 1 mM NaF; 5 mM EDTA; 1 mM sodium orthovanadate; cocktail of protease inhibitors (Thermo Fisher Scientific, Loughborough, UK) at 4°C (Agouni et al., 2011). The cells were then scraped using a cell scraper to collect all the lysates in one corner of the well and then transferred

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into labelled 1.5 mL micro tubes (Eppendorf) using a 200 µL pipette. The cell lysates were then clarified by centrifugation at 4°C for 15 minutes at 14,000 g. The supernatant was carefully transferred without disturbing the pellet of cell debris, into new labelled 1.5 mL micro tubes. Cell lysates (samples) were assayed for protein quantification following procedure on section 2.7.2 and then stored at -20°C until further analysis.

2.7.2 Protein assay

Protein concentrations were determined using the colorimetric bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Loughborough, UK). BCA assay is based on the principle of reduction of Cu+2 to Cu+ by proteins in an alkaline environment (Olson and Markwell, 2007). The BCA assay primarily relies on two reactions; firstly, the peptide bonds in protein reduce copper (Cu+2) ions from the cupric sulfate to form cuprous cation (Cu+) (a temperature dependent reaction). Secondly, two molecules of bicinchoninic acid chelate with each Cu+ ion formed in the first reaction, forming a purple-coloured product that strongly absorbs light at a wavelength of 540- 562 nm. The bicinchoninic acid-Cu+ complex is influenced in protein samples by the presence of cysteine/cysteine, tyrosine, and tryptophan side chains. At higher temperatures (37 to 60°C), peptide bonds assist in the formation of the reaction product (purple coloured complex). The amount of purple colour produced (BCA/Cu+ complex) or reduced Cu+2 is proportional to the amount of protein present in the solution (Olson and Markwell, 2007).

Bovine serum albumin (BSA) standard curve samples (0.125, 0.25, 0.5, 0.75, 1, 1.5 and 2 mg/mL) were prepared in RIPA per the manufacturer‘s instructions (Thermo Fisher Scientific, Loughborough, UK). Then, 5 µL of samples and standards

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were added in duplicates into a 96-well plate. The blank used was RIPA buffer only. Then, 200 µL of mixture of Pierce™ BCA reagents A and B (Thermo Fisher Scientific, Loughborough, UK) at the ratio of 1:50, was added to each well containing standards, samples or blanks. The plate was then gently mixed for few seconds on a plate shaker. After incubation for 30 minutes at 37°C, a purple colour had developed in the wells. Then the amount of protein present in each well was quantified by measuring the absorption spectrum at 540 nm within one hour of incubation and by comparing with known concentrations of the BSA standard curve. Briefly, a standard curve was constructed using Microsoft Excell and the protein concentrations were calculated using the standard curve obtained. The desired coefficient of determination (R2) was always over 0.96.

2.7.3. Sample preparation and protein denaturation for electrophoresis.

Protein samples were prepared for gel electrophoresis by mixing them in a 1:1 ratio with loading buffer containing 250 mM Tris-HCl pH 6.8, 10% sodium dodecyl sulfate (SDS), 50% glycerol and 0.025% bromophenol blue. SDS is an anionic detergent which denatures secondary and non–disulphide–linked tertiary structures, and applies a negative charge to each protein in proportion to its mass. Glycerol is added to increase the density of the samples to layer the samples at the bottom of the gels, and 50% bromophenol blue is a tracking dye to monitor protein migration. Then samples were heated to 95°C (6 minutes) in a heating block to further promote protein denaturation and help SDS to bind. Samples were then processed for western blotting or stored at -20°C until further analysis.

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2.7.4. Western blotting analysis.

After protein concentration and sample preparation as previously outlined, equivalent amounts of protein (20-25 μg) per sample were resolved by SDS-polyacrylamide gels (4-12%) (Sigma-Aldrich, Gillingham UK) for 1 hour 30 minutes at 100 volts in a TruPAGE™ TEA-Tricine SDS running buffer (Sigma-Aldrich, Gillingham UK) and transferred to a Immobilon-FL polyvinylidene difluoride (PVDF) transfer membrane (Merk Millipore®, Tullagreen, Germany) pre-soaked in methanol to activate the membrane. A wet transfer method was used to transfer proteins form the gel to the PVDF membrane for 2 hours at 70 volts or at 35 volts overnight (16 hours) at 4ºC in a NuPAGE® transfer buffer (Novex by Life Technologies, USA). The membranes were then blocked in Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBS-T) and 0.1% (w/v) non-fat dry milk at room temperature with regular shaking for 1 hour. Blot membranes were washed four times with TBS-T, 10 minutes for each wash. Membranes were then incubated overnight at 4°C with regular shaking with the appropriate dilution of the following primary antibodies: polyclonal rabbit anti-BiP at 1:500 dilution, monoclonal rabbit anti-phospho-eIF2α (S473) at 1:1,000 dilution, polyclonal rabbit anti- eIF2α at 1:1000, monoclonal mouse anti-eNOS antibody at 1:300 dilution, polyclonal rabbit anti-caveolin-1 at 1:300 dilution, monoclonal rabbit anti-phospho-eNOS at 1:500 dilution, mouse, polyclonal rabbit anti-VIMP (SelS) at 1:500 (Cell Signalling Technology, Boston, USA) and mouse anti-β-actin diluted at 1:5,000 (Santa Cruz, UK) were used as a loading controls. All primary antibodies recognize human proteins.

The membranes were then washed at least three times for 10 minutes per wash in TBS-T and incubated for 1 hour at room temperature with the appropriate fluorophore conjugated goat anti-mouse or goat anti-rabbit antibodies 1:1,5000

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(LICOR, UK). Immuno-reactivity was visualized with fluorophore conjugated secondary antibodies were detected immediately after washing with ODYSSEY®CLx infrared imaging system scanner (LI-COR Biotechnology - UK Ltd, Cambridge, UK). Each experiment was repeated at least three times.